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This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.
CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.
Single-nucleotide variants (SNVs) that cause missense or nonsense mutations are the most common source of mutations in the human genome1. To determine whether a particular SNV is pathogenic, and to shed light on its pathogenesis, precise animal models are required2. Zebrafish are good human disease models, exhibiting a high degree of physiological and genetic homology with humans, a short developmental cycle, and strong reproductive ability, which is advantageous for research into pathogenic characteristics and mechanisms, as well as drug screening3.
The clustered regul....
This study was conducted in strict compliance with the guidelines of the Care and Use Committee of the South China Normal University.
1. Preparing synthetically modified gRNA and zSpRY-ABE8e mRNA
The mutation of TSR2 has been reported to cause Diamond Blackfan anemia (DBA)42. Here, a DBA zebrafish model was constructed with a tsr2 (M1V) mutation using the i-Silence strategy. The adenine of the start codon of the zebrafish tsr2 was successfully converted to guanine using zSpRY-ABE8e (Figure 3).
The EditR analysis of the Sanger sequencing results showed that there was an A/G overlap at the adenine base of th.......
This protocol describes the construction of a zebrafish disease model using the base editor zSpRY-ABE8e. Compared with the traditional HDR pathway for base substitution, this protocol can achieve more efficient base editing and reduce the occurrence of indels. At the same time, this protocol involves implementing the recently proposed i-Silence gene-knockout strategy in zebrafish. Taken together, zSpRY-ABE8e will enhance the application of zebrafish models in disease research.
Off-target effec.......
We thank Barbara Garbers, PhD, from Liwen Bianji (Edanz) for editing the English text of a draft of this manuscript. This work was supported by the Key-Area Research and Development Program of Guangdong Province (2019B030335001), the National Key R&D Program of China (2019YFE0106700), the National Natural Science Foundation of China (32070819, 31970782), and the Research Fund Program of Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals (PBEA2020YB05).
....Name | Company | Catalog Number | Comments |
Agarose | Sigma-Aldrich | A9539 | 1.5% Agarose used to make an injection plate |
Borosilicate Glass Capillaries | Harvard Apparatus | BS4 30-0016 | |
Cell culture dishes | Falcon | 351029 | |
ClonExpress Ultra One Step Cloning Kit | Vazyme | C115 | Kit for Infusion clone |
Codon optimization service | Sangon Biotech | ||
Drummond Microcaps | Drummond Microcaps | P1299-1PAK | Length:32 mm, capacity:0.5 μL |
EasyEdit gRNA service | GenScript | ||
Fine Forceps | Fine Scientific Instrument | 11254-20 | Used to break meedle |
Flaming/Brown Micropipette Puller | Stutter Instrument | P-97 | Used to pull the glass capillaries |
HotStart Taq PCR StarMix | Genstar | A033-101 | Used for PCR reaction |
Intelligent artificial climate box | TENLIN | PRX-1000A | Used to culture zebrafish embryos |
Methylcellulose | Sigma-Aldrich | M0512 | Used to fix zebrafish when photographing |
Microloader pipette tips | Eppendorf | 5242956003 | |
mMACHINE kit | |||
Mut Express II Fast Mutagenesis Kit V2 | Vazyme | C214-01 | Kit for site-directed mutagenesis |
Pneumatic Microinjector | ZGene Biotech | ZGPCP-1500 PLUS | |
pT3TS-zSpCas9 | Addgene | 46757 | |
RNeasy FFPE kit | Qiagen | 73504 | Kit for RNA purification |
Sanger Sequencing service | Sangon Biotech | ||
Sodium hydroxide, granular | Sangon | A100173-0500 | NaOH used for genome extraction |
Stereo Microscope | Olympus | SZX10 | Used for photograph of phenotype |
SZ Series Zoom Stereo Microscope | CNOPTEC | SZ650 | |
T3 mMESSAGE | Ambion | AM1348 | Kit for in vitro transcription |
TIANprep Mini Plasmid Kit | TIANGEN | DP103-03 | Kit for plasmid extraction kit |
TIANquick Mini Purification Kit | TIANGEN | DP203-02 | Kit for purification for linearized plasmid |
Tricaine | Sigma-Aldrich | E10521 | Used to anesthetize zebrafish |
Tris (hydroxymethyl) aminomethane | Sangon | A600194-0500 | Component of Tris·HCl used for genome extraction |
XbaI | New England Biolabs | R0145S | Restriction endonuclease used for plasmid linearization |
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